Use of chromobacterium substugage formulations, compositions and compounds to modulate cornworm rootworm larvae infestation

ABSTRACT

Provided is the use of or compositions or formulations comprising  Chromobacterium  species, filtrate, supernatant, extract, pesticidally active compound or metabolite derived therefrom as an insecticide, particularly against infestation of Corn Rootworm larvae.

The instant application is a divisional of U.S. Ser. No. 13/842,981 filed on Mar. 15, 2014.

TECHNICAL FIELD

Provided is the use of or compositions or formulations comprising Chromobacterium species, filtrate, supernatant, extract, pesticidally active compound or metabolite derived therefrom as an insecticide, particularly against infestation of Diabrotica (Corn Rootworm) larvae.

BACKGROUND

In 2000, Dr. Martin and her coworkers at USDA isolated a purple-pigmented bacteria (PRAA4-1) from a forest soil in Maryland (Martin et al., 2007a). It is a facultatively aerobic, motile, Gram-negative betaproteobacterium with polar flagella. Colonies formed at 2-3 days on an L-agar plate at 25° C. are initially cream colored, gradually turning light to dark violet during the following 24 hours. Colonies of PRAA4-1 grow well on peptone based media with an optimum at 25° C., pH 6.5-8.0, and with 0-1.5% (w/v) NaCl (Martin et al., 2007a). This motile, Gram-negative, bacteria was identified as a new species of Chromobacteria, Chromobacterium substsugae sp. nov (Martin et al., 2007c). and or alternatively as Chromobacterium substsugae NRRL B-30655.

The effect of Chromobacterium substsugae NRRL B-30655 on insects does vary. It has been found to be toxic to Colorado Potato Beetle larvae but not to adults when these insects were fed a diet including Chromobacterium substsugae NRRL B-30655 (Martin, 2007b, 2007c). It was found to be toxic to adult southern green stink bugs but appeared to have a faster effect on males (Martin 2007c). For diamondback moth instar larvae fed a diet including Chromobacterium substsugae NRRL B-30655, the mortality was 90% in 7 days. None of the gypsy moth larvae died following treatment with NRRL B-30655, but the larvae which consumed NRRL B-30655 in their diet were 40% lighter than the controls. For mosquito larvae, there was no mortality at 48 hrs. although the larvae in the B. thuringiensis control were dead in 16 hrs.

With respect to Southern and Western Corn Rootworms, about 80% of both Southern and Western Corn Rootworm adults died when fed Chromobacterium substsugae NRRL B-30655 in a bait formulation; in other assays 100% mortality was reached after 120 hr. (Martin, 2007c). However, mortality in southern corn rootworm larvae was only found to be 40% when fed whole cultures. There was though a difference in the weight in larvae fed NRRL B-306555 as compared to the controls (Martin, 2007c).

US patent application publication no. US20120100236 also discloses compounds obtainable or derived from Chromobacterium species, more particularly, Chromobacterium substugae. In particular, PCT appln. no. PCT/US2012/061503 discloses the use of Chromobacterium species as an acaricide and insecticide, particularly against infestation of one or more pests belonging to the Acarina, Scarabeidae, Drosophilidae, Triozidae, Aphidae, Muscidae, Anthomyiidae or Tenebrionidae families.

SUMMARY

Provided is a method for modulating infestation of corn rootworm larvae in a location where modulation is desired comprising applying an amount of (a) a culture, suspension or whole cell broth comprising a strain of Chromobacterium sp., or supernatant, filtrate, cell fraction, extract and/or one or more compounds derived from said culture, suspension or whole cell broth and (b) optionally at least one of a carrier, diluent or adjuvant effective to modulate said infestation of corn rootworm larvae at said location. In a particular embodiment, the strain has the identifying characteristics of Chromobacterium substugae NRRL B-30655.

Infestation of Diabrotica (corn rootworm) larvae may be modulated by modulating mortality of corn rootworm larvae, specifically by modulating and particularly increasing or boosting mortality of corn rootworm larvae and/or by decreasing hatching rate of eggs laid and/or decreasing the number of eggs laid in a particular location.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the mean percent (%) mortality of corn rootworm first instar larvae on MBI-203 treated corn seedlings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While the compositions and methods heretofore are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

As defined herein, “derived from” means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source. In the event that the “source” is an organism, “derived from” means that it may be isolated or obtained from the organism itself or culture broth, suspension or medium used to culture or grow said organism. A compound “derived from” or “obtainable from” means that a compound may be isolated from or produced by a cell culture, whole cell broth, suspension, filtrate, supernatant, fraction or extract.

As defined herein, “whole broth culture” or “whole cell broth” refers to a liquid culture containing both cells and media. If bacteria are grown on a plate, the cells can be harvested in water or other liquid, whole culture. The terms “whole broth culture” and “whole cell broth” are used interchangeably.

As defined herein, “supernatant” refers to the liquid remaining when cells grown in broth or are harvested in another liquid from an agar plate and are removed by centrifugation, filtration, sedimentation, or other means well known in the art.

As defined herein, “filtrate” refers to liquid from a whole broth culture that has passed through a membrane.

As defined herein, “extract” refers to liquid substance removed from cells by a solvent (water, detergent, buffer, organic solvent) and separated from the cells by centrifugation, filtration or other method.

As defined herein, “metabolite” refers to a compound, substance or byproduct of a fermentation of a microorganism, or supernatant, filtrate, or extract obtained from a microorganism that has pesticidal activity.

As defined herein, an “isolated compound” is essentially free of other compounds or substances, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by analytical methods, including but not limited to chromatographic methods, electrophoretic methods. A compound “derived from” a Chromobacterium species also encompasses a metabolite.

As defined herein, “carrier” is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to plant or other object to be treated, or its storage, transport and/or handling.

The term “diluent” is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the active ingredient.

As defined herein, “modulate”, is used to mean to alter the amount or rate of pest infestation.

As defined herein, “pest infestation”, is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired weed in a growth system.

As defined herein “pesticide”, is a substance derived from a biological product or chemical substance that increase mortality or inhibit the growth rate of plant pests and includes but is not limited to nematicides, algaecides, herbicides, insecticides, plant fungicides, plant bactericides, and plant viricides.

Methods of Production As noted above, the pesticide used in the method set forth herein may comprise or be derived from an organism having the identifying characteristics of a Chromobacterium species, more particularly, from an organism having the identifying characteristics of a strain of Chromobacterium substugae, more particularly from a strain of Chromobacterium substugae sp. nov. which may have the identifying characteristics of NRRL B-30655, or alternatively from any other microorganism. The methods comprise cultivating these organisms and obtaining the compounds and/or compositions of the present invention by isolating these compounds from the culture of these organisms.

In particular, the organisms are cultivated in nutrient medium using methods known in the art. The organisms may be cultivated by shake flask cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in suitable medium and under conditions allowing cell growth. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available may be available from commercial sources or prepared according to published compositions.

After cultivation, the compounds, metabolites and/or compositions may be extracted from the culture broth. The extract may be fractionated by chromatography. Compounds used may be metabolites and in a specific embodiment may include but is not limited to compounds set forth in US Patent Application Publication No. US20120100236 and PCT appln. no. PCT/US2012/061503, the contents of both which are incorporated herein by reference.

Compositions

The substances set forth above used in the compositions and methods disclosed herein can be formulated in any manner. Non-limiting formulation examples include but are not limited to Emulsifiable concentrates (EC), Wettable powders (WP), soluble liquids (SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders (SP), Microencapsulation, Water dispersed Granules, Flowables (FL), Microemulsions (ME), Nano-emulsions (NE), and Seed treatments, etc. In any formulation described herein, percent of the active ingredient is within a range of 0.01% to 99.99%.

The compositions may be in the form of a liquid, gel or solid. Liquid compositions comprise pesticidal compounds derived from a Chromobacterium strain, e.g. a strain having the identifying characteristics of Chromobacterium substugae sp. Nov and more particularly, having the identifying characteristics of NRRL B-30655 (see U.S. Pat. No. 7,244,607).

A solid composition can be prepared by suspending a solid carrier in a solution of pesticidal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65° C. or lower.

A composition may comprise gel-encapsulated compounds derived from the Chromobacterium strain. Such gel-encapsulated materials can be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated Chromobacterium, or a cell-free filtrate or cell fraction of a Chromobacterium culture or suspension, or a spray- or freeze-dried culture, cell, or cell fraction or in a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.

The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA Inerts List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of surfactants may range between 0.1-35% of the total formulation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ionic, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of the composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.

The composition as set forth above also comprises a stabilizing agent, which stabilizes a biological pesticide composition against physical separation and loss of activity due to exposure to sunlight. This stabilizing agent may be a benzoic acid salt or lignin sulfonate salt.

The composition may as noted above, further comprise an insecticide. The insecticide may include but is not limited to avermectin, Bt, neem oil, spinosads, Burkholderdia sp. as set forth in US Patent Appin. Pub. No. 2011-0207604, entomopathogenic fungi such a Beauveria bassiana and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, and neonicotinoids.

Uses

The compositions, cultures and supernatants and pesticidal compounds set forth above may be used particularly to modulate infestation of corn rootworm larvae on a plant, plant seed, plant part, plant roots. seedling or substrate (e.g., soil, sand, loam, clay) for growing said plant, particularly a corn plant, cucurbits (e.g., cucumbers, melons, pumpkins, squash, gourds, etc.), wheat, barley, oats, rye, sorghum, beans and legumes, peas, potato, sweet potato, soy, oilseed rape, tomato, aubergine, lettuce, pepper, sunflower, and ornamental plants such as chrysanthemum as well as other plants such as red root pigweed (Amaranthus retroflexus), goosegrass (Eleusine indica) weeping lovegrass (Eragrostis curvula), sand lovegrass (Eragrostis trichodes), rhodes grass (Chloris gayana), shattercane (Sorghum drummondii), johnsongrass (Sorghum halepense), maize (Zea mays), sandbur (Cenchrus tribuloides), large crabgrass (Digitaria sanquinalis), barnyard grass, (Echinochloa crus-galli), woolly cupgrass (Eriochloa villosa), witchgrass (Panicum capillare), foxtail millet (P. italicum), Prosso millet (P. miliaceum), switchgrass (P. virgatum), giant foxtail (Setaria faberi), yellow foxtail (S. pumila), bristly foxtail (S. vericillatta), Green foxtail (S. viridis), texas panicum (Urochloa texana), Redtop (Agrostis gigantean), oat (Avena sativa), reed canarygrass (Phalaris arundinacea), downy brome (Bromus tectorum), Orchardgrass (Dactylis glomerata), western wheatgrass (Pascopyrum smithii), spring wheat “Russ” (Triticum aestivum)

It may be used to modulate members go the genus Diabrotica (rootworm) including Diabrotica balteata (banded cucumber beetle), Diabrotica barberi (northern corn rootworm), Diabrotica beniensis, Diabrotica cristata, Diabrotica curvipustulata, Diabrotica dissimilis, Diabrotica elegantula, Diabrotica emorsitans, Diabrotica graminea, Diabrotica hispanolae, Diabrotica lemniscata, Diabrotica linsleyi, Diabrotica longicornis, Diabrotica milleri, Diabrotica nummularis, Diabrotica occlusa, Diabrotica porracea, Diabrotica scutellata, Diabrotica speciosa (cucurbit beetle or chrysanthemum beetle), Diabrotica tibialis, Diabrotica trifasciata, Diabrotica undecimpunctatahowardi (southern corn rootworm, a.k.a spotted cucumber bettle), Diabrotica undecimpunctata tenella (western cucumber beetle), Diabrotica undecimpunctata undecimpunctata (western spotted cucumber beetle), Diabrotica virgifera virgifera (western corn rootworm), Diabrotica virgifera zea (Mexican corn rootworm), Diabrotica viridula, Diabrotica significata (three-spotted cucumber beetle)

Southern, Western or Northern Corn Rootworm larvae. In a particular embodiment, infestation of corn rootworm larvae is modulated by modulating mean mortality of said larvae at a particular location, particularly at least about 50% and more particularly at least about 60% within at least about three days following application of said compositions, cultures and supernatants and pesticidal compounds set forth above.

EXAMPLE

The composition and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.

Methods

A bioassay set up was done to determine the effect of a formulation of Chromobacterium substugae sp. nov. (MBI-203) on Southern Corn Rootworm larvae. Newly hatched corn rootworm larvae were used in the bioassay.

Corn seeds were soaked in water overnight for germination. After soaking, the water-imbibed seeds were laid out in a tray lined with moistened paper towels (at least 4 layers of paper towels were used), with seeds spread evenly in the tray. The seeds were then covered with 2-3 layers of paper towels. The paper towel on top of corn seeds was moistened with water until paper towels are completely wet, just enough to not to flood the seeds with water. Seeds in wet paper towels were then covered with a plastic wrap to contain high humidity and kept at room temperature. The seeds were allowed to germinate for 2-3 days.

Bioassay setup was done using 100 mm×15 mm sterile plastic petri dishes. Petri dishes were lined with 4 layers of paper towels moistened with deionized water. Five germinated corn seedlings were selected and placed in petri dishes. Bioassay setup was done with 4 treatments;

a) MBI 203 TGAI (technical grade of active ingredient) at 3% vol:vol concentration, b) MBI-203 TGAI (technical grade of active ingredient) at 6% vol:vol concentration, c) MBI-203 TGAI at 3% vol:vol concentration, and d) dH₂O (negative control). Each treatment replicated three times. Petri dishes with corn seedlings were labeled according to treatment. Treatment of seedlings was done by spraying 1 mL of treatment solution on seedlings in each dish using a spray bottle. Spray application was done in fume hood and allowed to air dry. After drying, 10 first instar corn rootworm larvae were introduced to each dish with treated corn seedlings. The seedlings in each dish were covered with moist paper towel before covering with the dish cover. The dishes with corn rootworm larvae were kept at room temperature. Larval mortality was assessed 3 days post introduction to treated seedlings. Results

The MBI 203 TGAI at different concentration affected corn rootworm larvae 3 days post exposure to treated corn seedlings. Corn rootworms were found susceptible to the product with a 60% mean mortality attained on corn seedlings treated with 10% MBI 203 TGAI. MBI 203 concentrations at 3% and 6% caused 53.3% and 50% mortality (FIG. 1). All treatments have showed 50% or more larval mortality after 3 days of exposure.

Although this invention has been described with reference to specific embodiments, the details thereof are not to be construed as limiting, as it is obvious that one can use various equivalents, changes and modifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

REFERENCES

-   Clark, T L and B E Hibbard. 2004. Comparison of Nonmaize Hosts to     Support Western Corn Rootworm (Coleoptera:Chrysomelidae) Larval     Biology. Environ. Entomol. 33(3): 681-689. -   Martin, P. A. W., D. Gundersen-Rindal, et al. (2007a).     “Chromobacterium substugae sp. nov., a betaproteobacterium toxic to     Colorado potato beetle and other insect pests.” Int. J. Syst. Evol.     Microbiol. 57: 993-999. -   Martin, P. A., A .D. S. Shropshire, et al., (2007b).     “Chromobacterium substugae sp. nov. for control of insect pests”     U.S. Pat. No. 7,244,607 B2. -   Martin, P. A. W., Hirose, E., and Aldrich, J. R. 2007c. “Toxicity of     Chromobacterium substugae to southern green stink bug     (Heteroptera:Pentatomidae) and corn rootworm     (Coleoptera:Chrysomelidae)”. J. Econ. Entomol. 100: 680-684. -   Martin, P. A. W., Blackburn, M., et al. (2004), “Two New Bacterial     Pathogens of Colorado Potato Beetle (Colorado:Chrysomelidae)”, J.     Econ. Entomol. 97:774-780 (2004). 

What is claimed is:
 1. A method for inhibiting infestation of Diabrotica (corn rootworm) larvae in a location where inhibition is desired comprising the steps of: applying an amount of a whole cell broth comprising a strain of Chromobacterium sp., effective to inhibit said infestation of corn rootworm larvae at said location by inducing mortality in at least 50% of said corn rootworm larvae in said location.
 2. The method according to claim 1, wherein the location where inhibition is desired is on a plant, plant seed, plant roots, plant part, seedling or a substrate for growing said plant.
 3. The method according to claim 1, wherein said Chromobacterium sp. is Chromobacterium subtsugae sp. Nov strain.
 4. The method according to claim 1 wherein said Diabrotica (corn rootworm) larvae are Southern Corn Rootworm larvae.
 5. The method according to claim 1, which further comprises applying another insecticidal substance.
 6. A method for inhibiting infestation of Diabrotica (corn rootworm) larvae in a location where inhibition is desired comprising the steps of: applying an amount of a culture, a suspension, a supernatant, a filtrate, a cell fraction, or an extract comprising a strain of Chromobacterium sp., effective to inhibit said infestation of corn rootworm larvae at said location by inducing mortality in at least 50% of said corn rootworm larvae in said location, wherein the location where inhibition is desired is on a plant, plant seed, plant roots, plant part, seedling or a substrate for growing said plant.
 7. The method according to claim 6, wherein said Chromobacterium sp. is Chromobacterium subtsugae sp. nov strain.
 8. The method according to claim 6, wherein said Diabrotica (corn rootworm) larvae are Southern Corn Rootworm larvae. 